Liposome-encapsulated poly ICLC

ABSTRACT

Although poly ICLC possess a broad spectrum of antimicrobial and anticancer activities, it therapeutic potential has yet to be fulfilled due to its toxic side effect. This problem can be overcome by encapsulating poly ICLC within liposomes which provides a drug delivery system with slow sustained release characteristic and which has the ability to target the drug to sites of infection and tumor without causing systemic burden to normal tissues, thereby enhancing the immunological and biological activities of poly ICLC.

FIELD OF THE INVENTION

[0001] The present invention relates to a poly ICLC formulation withimproved therapeutic efficacy.

BACKGROUND OF THE INVENTION

[0002] Double-stranded RNAs (dsRNAs) are very potent biologic modifiers.They can exert a profound influence on cells at nanomolarconcentrations. The modulating effects of dsRNA include a broad spectrumof actions at the molecular and cellular levels. At the molecular level,dsRNAs can elicit biological effects such as interferon synthesis,induction of protein kinase, induction of 2-5A polymerase, enhancementof histocompatibility antigen and inhibition of metabolism. And at thecellular level, dsRNA can elicit biological effects such aspyrogenicity, mitogenicity, macrophage activation, activation ofcell-mediated immunity and induction of antiviral state. One promisingpotential of dsRNAs is its immunomodulating effect in antimicrobial andanticancer therapies. In particular, the double-stranded RNA poly ICLC,or PICLC for short, was found highly effective as an antiviral orantitumor agent.

[0003] Poly ICLC is a synthetic dsRNA consisting of polyriboinosinic andpolyribocytidylic acid strands (poly I.poly C) stabilized withpoly-L-lysine and carboxymethylcellulose. The resulting poly ICLC isthermodynamically more stable than poly I.poly C. Poly ICLC has beenshown in clinical trials to be effective in the cancer treatment ofgliomas (Salazar, A. M., & al., Neurosurgery 38:1096-1104). It has alsobeen shown in a number of studies to be effective in the immunotherapyof viral infection including influenza (Wong. J. P., Antimicrob. AgentsChemother. 39:2574-2576), rabies (Baer, G. M., J. Infect. Dis.136:286-292), Rift Valley fever (Kende, M., J. Biol. Response Modifiers4:503-511) and Venequelan equine encephamyelitis (Stephen, E. L., J.Infect. Dis. 136:267-272).

[0004] Although poly ICLC is a promising immunomodulator which has greatpotential in antimicrobial and anticancer therapies, it has been shownto produce serious side effects in humans, especially when the drug isadministered in multiple high doses. Some of the reported side effects(Levine, A. S., Cancer Treat. Rep. 62:1907-1913) include fever,hypotension, leukopenia, myalgia, thrombocytopenia and poly arthalgia.The inherent toxicity problem must be overcome to render poly ICLC saferfor use in humans. Furthermore, the therapeutic efficacy of poly ICLC islimited by its stability in vivo. As a ribonucleic acid, poly ICLC issusceptible to degradation in the body by serum RNAse. Although theextent of RNAse degradation of poly ICLC is much improved as compare tothat of poly I.poly C, the protection is not complete and poly-L-lysineand carboxymethylcellulose themselves may be susceptible to enzymaticdegradation and immunological clearance in vivo. Therefore, a needexists for an improved formulation of poly ICLC which has improvedtherapeutic efficacy and will be safer for use in humans.

SUMMARY OF THE INVENTION

[0005] It is an object of the present invention to provide a poly ICLCformulation having enhanced therapeutic efficacy while reducing it toxiceffect in humans.

[0006] In accordance with one aspect of the present invention, there isprovided an immunomodulating agent comprising poly ICLC encapsulatedwithin liposomes. Preferably, the liposomes used are unilamellar ormultilamellar and contain at least one cationic phospholipid such asstearylamine, 1,2-diacyl-3-trimethylammonium-propane (TAP) or1,2-triacyl-3-dimethylammonium-propane (DAP). Most preferably, theliposomes are unilamellar or multilamellar liposomes prepared from thelipids phosphatidylcholine and stearylamine, and the steroid cholesterolat a molar ratio of approximately 9:1:1, respectively. The surfaceliposomes may be coated with polyethylene glycol to prolong thecirculating half-life of the liposomes, and with antibody for targetingto specific sites in the body.

[0007] Neutrally charged liposomes can also be used for liposomalentrapment of poly ICLC. Such neutrally charged liposomes can beprepared by using, for example phosphatidylcholine and cholesterol.

[0008] In accordance with another aspect of the present invention thereis provided a method for preparing liposomal poly ICLC comprising thestep of freeze-drying a mixture of liposomes and poly ICLC. Convenientlythe method includes removing organic solvent from a mixture ofphospholipids, rehydrating the resulting lipids mixture with an aqueousbuffer containing poly ICLC, freeze-drying the resulting lipid-poly ICLCmixture, reconstituting the resulting dried mixture, and resuspendingthe resulting liposome pellets with a buffer solution to the desireddrug concentration prior to use. Suitable buffer can be phosphatebuffered saline, normal saline or deionized water. It is important forthe preparation of buffer solution to use RNAse-free water so thatenzymatic degradation of poly ICLC can be minimized.

[0009] Alternate methods of preparation of liposomes include detergentdialysis, extrusion, reverse-phase evaporation (REV) and sonication. Theloading of poly ICLC into the liposomes can be achieved by passivetrapping and by active process such as remote loading. The unentrappedpoly ICLC can be removed by centrifugation, column separation or bydialysis.

[0010] The advantages of encapsulating poly ICLC in liposomes are thatthe toxicity of poly ICLC is decreased, and at the same time thetherapeutic efficacy of poly ICLC is increased. Furthermore, liposomalpoly ICLC protects the poly ICLC from RNAse degradation in the body,thereby enhancing the immunological and biological activities of polyICLC.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011]FIG. 1 is a graph showing results of tests relating to thetherapeutic efficacy of free poly ICLC versus that of liposomal polyICLC.

[0012]FIG. 2 is a graph showing the results of tests relating to thetoxicity of free poly ICLC versus that of liposomal poly ICLC.

DETAILED DESCRIPTION

[0013] Poly ICLC

[0014] Poly ICLC was prepared by the Pharmaceutical Services, College ofPharmacy University Of Iowa (Iowa City, Iowa), and was provided by theNational Institute of Health (Bethesda, Md.). Each milliliter of polyICLC contained 2 mg poly I.poly C, 1.5 mg poly-L-lysine, and 5 mgcarboxymethylcellulose in 0.9% sodium chloride.

[0015] Encapsulated-liposome Poly ICLC

[0016] Liposomes are microscopic lipid vesicles consisting of one ormore lipid bilayer(s) and aqueous compartment(s). The primaryconstituents of liposomes are usually a combination of phospholipids andsteroid, such as cholesterol. The phospholipids can be positively,neutrally and negatively charged. Liposomes made from positively andnegatively charged phospholipids are called cationic and anionicliposomes, respectively. DNA and RNA are usually negatively charged,therefore, cationic liposomes are the liposomes of choice for makingliposomal poly ICLC formulation. The cationic phospholipid used formaking liposomal poly ICLC is preferably stearylamine,1,2-diacyl-3-trimethylammonium-propane (TAP) or1,2-triacyl-3-dimethylammonium-propane (DAP). Cholesterol is includedfor stabilization of the bilayer. The surface liposomes may be coatedwith polyethylene glycol to prolong circulation thereof.

[0017] Proteins can also be combined with the liposome membranes topromote binding with specific cell receptors. Liposomes used forentrapment of poly ICLC may be large multilamellar vesicles (MLVs),small unilamellar vesicles (SUVs) or large unilamellar vesicles (LUVs).Preferably, MLVs are used for preparing liposomal poly ICLC.

[0018] When used as a drug delivery system, liposomes are known to havea slow sustained release characteristic and the ability to target drugsto sites of infection and tumor without causing systemic burden tonormal tissues. Liposomes have been used successfully to entrap a numberof therapeutic drugs, including antibiotics, antivirals, and anticancer.Because of these attributes, liposomal poly ICLC is an excellent drugdelivery system which can significantly decrease the dose-relatedtoxicity of poly ICLC. Furthermore, liposome-encapsulation protects thepoly ICLC from RNAse degradation in the body, thereby enhancing thetherapeutic efficacy of poly ICLC.

[0019] Preparation

[0020] The liposomes were prepared using 210 mg of phosphatidylcholine(210 μmole), 23.2 mg stearylamine (23.2 μmole) and 8.1 mg cholesterol(30 μmole). The lipids were added in a 100 ml round bottom flask, 2 mlof chloroform was added to dissolve the lipids. The round bottom flaskwas rotary evaporated in a 45° C. water bath until a dried lipid filmwas formed. The flask was then placed in a vacuum oven (45° C., −80 Kpa)for one hour to remove residual organic solvent. The lipid film was thenreconstituted with 3 ml of poly ICLC (2 mg/ml) followed by 3 ml of 0.9%NaCl. Other suitable buffers can be phosphate buffered saline, normalsaline or deionized water. It is important for the preparation of buffersolution to use RNAse-free water to minimize degradation of poly ICLC.The lipid-drug mixture was then transferred to a screwcapped tube, mixedwell, and frozen by immersing the tube in liquid nitrogen. The samplewas then lyophilized overnight until all the liquid was removed toobtain a white dried powder. Following lyophilization, the sample wasrehydrated with 100-150 μl 0.9% NaCl, incubated for 15 min. at 45° C.,and left undisturbed for 2 hr. at room temperature. The liposomal polyICLC was diluted in sterile 0.9% NaCl and washed using anultracentrifugation step. The liposome pellet was then resuspended witha buffer solution to the desired drug concentration for administrationinto mice.

[0021] The surface of the liposomes may be coated withpolyethyleneglycol to prolong circulation and with an antibody toincrease the affinity of the liposome to specific sites of infection andtumor.

[0022] Neutrally charged liposomes can also be used for liposomalentrapment of poly ICLC. For example, the neutrally charged liposomescan be prepared using phosphatidylcholine and cholesterol.

[0023] Other methods of preparation to produce liposomes includedetergent dialysis, extrusion, reverse-phase evaporation (REV) andsonication. The loading of poly ICLC into the liposomes can be achievedby passive trapping or by active process, such as remote loading. Theunentrapped poly ICLC can be removed by centrifugation, columnseparation or by dialysis.

[0024] Adaptation of Egg-propagated Influenza A/PR/8 Virus in Mice

[0025] Using conventional procedures, influenza A/PR/8 virus wascommunicated to mice through lung passages by four blind passagesutilizing egg-propagated virus (available from ATTC, Parklawn, Md.) asthe initial inoculum. The virus became pathogenic in mice as early asthe third passage. The symptoms of influenza were standing fur, rapidloss of body weight, grouping together and significant loss of animal'smovement inside the cages. Post-mortem examination of the infected micerevealed severe pulmonary lesions and pulmonary enlargement was alsoobserved in some mice.

[0026] Testing

[0027] Liposome-encapsulated poly ICLC was administered to the mice byintranasal, intraperitoneal or intravenous routes. The volumes ofinoculum used were 50 μl for the intranasal route and 100 μl by theintraperitoneal and intravenous routes. For the intranasal andintraperitoneal routes, mice were anaesthetized with sodiumpentobarbital prior to administration of the drug. When the animals wereunconscious, they were carefully supported by hands with their nose up,and the antiviral agents were gently applied with a micropipette intothe nostrils. The applied volume was naturally inhaled into the lungs.

[0028] Groups of anesthetized mice (5-10 mice per group) were given oneor two doses (20 μg/dose) of poly ICLC or liposome-encapsulated polyICLC by the intraperitoneal or intravenous route. The doses were givento the mice 7, 14 and 21 days prior to virus challenge. The mice werethen intranasally infected with 10 LD₅₀ mouse-adapted influenza A/PR/8virus. At day 14 post virus infection, the number of mice which survivedthe virus challenged was recorded.

[0029] Results

[0030] The efficacy of free and liposome-encapsulated poly ICLC for theprophylactic protection of mice against lethal challenges of influenza Ainfection in mice is shown in FIG. 1. In comparison, mice which wereadministered free poly ICLC within 7 days prior to virus infection had a100% survival rate at day 14 post virus infection. However whenpretreatment of free poly ICLC were given at days 14 and 21 prior tovirus challenge, the survival rates at day 14 post infection decreased.In contrast, mice which were given liposome-encapsulated poly ICLC (MLVpoly ICLC) within days 7 and 14 prior to virus challenge had a 100%survival rate at day 14 post virus infection. These results showed thatliposome encapsulation did not adversely affect the antiviral andimmunomodulating activities of poly ICLC, but, rather enhanced theseactivities by prolonging the antiviral state.

[0031] Referring now to FIG. 2, there is shown the effect of toxicity offree and liposomal poly ICLC on mice as measured by their body weight.Mice which have a toxic dose of poly ICLC will experience signs, such asrapid loss in body weight, piloerection and decreased body movement.Mice were administered two daily doses of 30 μg/animal of free polyICLC. Referring to FIG. 2, the first dose was given at day −2 post drugadministration and the second dose was given at day 0 post drugadministration. It was found that mice were loosing up to 2 g (close to10% of total body weight) within 1-3 days after each administration. Inaddition to the loss of body weight, these mice also showed abnormalsymptoms or signs of piloerection (ruffled fur) and decreased bodymovement. In contrast, mice given identical doses of theliposome-encapsulated poly ICLC did not have significant loss of bodyweight, nor did they show any signs of piloerection and loss ofmovement. Therefore, it was found that free unencapsulated poly ICLC hadhigh toxicity, whereas liposome-encapsulated poly ICLC had a lowtoxicity as shown from the results in FIG. 2. The mice which wereadministered with liposomal poly ICLC did not exhibit a significant lossof body weight.

[0032] In conclusion, the results showed that free poly ICLC whenadministered directly into mice provided limited protection againstinfluenza A virus infection. Moreover, poly ICLC was shown to be verytoxic to mice. In contrast, liposome-encapsulated poly ICLC providedeffective treatment against viral infections by enhancing thetherapeutic efficacy while decreasing the toxicity of poly ICLC.

We claim:
 1. An immunomodulating agent comprising poly ICLC encapsulatedwithin liposomes.
 2. An immunomodulating agent as claimed in claim 1wherein the liposomes include a mixture of phospholipids.
 3. Animmunomodulating agent as claimed in claim 2 wherein the mixture ofphospholipids includes at least one cationic phospholipid.
 4. Animmunomodulating agent as claimed in claim 3 wherein the cationicphospholipid is selected from the croup consisting of stearylamine,1,2-diacyl-3-trimethylammonium-propane and1,2-triacyl-3-dimethylammonium-propane.
 5. An immunomodulating agent asclaimed in claim 1 wherein the liposomes further include a steroid. 6.An immunomodulating agent as claimed in claim 5 wherein the steroid ischolesterol.
 7. An immunomodulating agent as claimed in claim 1 whereinthe liposomes are selected frorr.